Determination of the labile iron pool of human lymphocytes using the fluorescent probe, CP655.

The present study introduces a method for determining the labile iron pool (LIP) in human lymphocytes. It is measured using the probe CP655, the fluorescence of which is stoichiometrically quenched by the addition of iron. The intracellular CP655 fluorescence in lymphocytes was quenched by increasing intracellular iron concentrations using the highly lipophilic 8-hydroxyquinoline iron complex. Intracellular fluorescence quenching, mediated by the physiological intracellular labile iron, can be recovered on the addition of excess membrane-permeable iron chelator, CP94. The intracellular probe concentration was measured using laser scanning microscopy. An ex situ calibration was performed in a "cytosolic" medium based on the determined intracellular CP655 concentration and probe fluorescence quenching in the presence of iron. The concentration of the LIP of healthy human lymphocytes was determined to be 0.57 +/- 0.27 microM. The use of the fluorescent probe CP655 renders it possible to record the time course of iron uptake and iron chelation by CP94 in single intact lymphocytes.


Introduction
In the normal individual, intracellular iron levels are under extremely tight control. However, when the iron status is changed, such as during periods of regular blood transfusion, iron in excess of functional requirements is present in the "labile iron pool" (LIP) which readily donates iron to ferritin. In principle, this loosely bound iron in living cells is able to redox-cycle between the two common oxidation states, thereby resulting in free radicals such as superoxide and the hydroxyl radical, which can damage biomolecules including DNA and lipids (Halliwell and Gutteridge, 1999). In order to quantify the LIP, a number of methods (Kozlov et al. 1992;Stuhne-Sekalec et al. 1992;Nielsen et al. 1993;Evans and Halliwell, 1994) including fl uorescence spectroscopy (Zanninelli et al. 2002;Breuer et al. 1995;Petrat et al. 1999Petrat et al. , 2000Stäubli and Boelsterli, 1998;Loyevsky et al. 1999;Kakhlon et al. 2001;Darbari et al. 2003) have been introduced. Although progress has been made in recent years towards the successful application of fl uorescent indicators for the determination of the intracellular labile iron pool in hepatocytes (Petrat et al. 1999(Petrat et al. , 2000Stäubli and Boelsterli, 1998) and erythrocytes (Loyevsky et al. 1999;Kakhlon et al. 2001;Darbari et al. 2003), the LIP of lymphocytes has been hardly studied. Lymphocytes are present in the systemic circulation and as such experience continual exposure to relatively high oxygen concentrations leaving the rise of iron-dependent free radical formation. Gackowski et al. 2002 have reported a method to measure the lymphocyte chelatable iron pool based on the fl uorescent probe calcein. However, there are several disadvantages associated with this method, namely its lack of selectivity and the requirement to use ester analogues in order to achieve suffi cient cellular uptake. We have recently reported the synthesis of several fl uorescent probes containing 3-hydroxypyridin-4-one (HPO), which have similar iron-binding properties to that of typical HPOs and contain a fl uorescent reporter group that undergoes quenching on interaction with iron (Ma et al. 2004(Ma et al. , 2005(Ma et al. , 2006, and found 7diethylamino-N-((5-hydroxy-6-methyl-4-oxo-1,4-dihydropyridin-3-yl)methyl)-N-methyl-2-oxo-2Hchromen-3-carboxamide (CP655) to be the most sensitive to the presence of iron. Therefore, in the present work we study the suitability of the HPO fl uorescent iron indicator CP655 to selectively chelate and quantify the labile iron pool of isolated human lymphocytes.

Isolation of lymphocytes
Blood (20 ml) from healthy donors (male members of the institute, 25-35 years old) was collected in two 10 ml sodium heparin preservative-free vacutainer tubes, immediately transferred to a 50 ml falcon tube and diluted with an equal volume (20 ml) of RPMI-1640 medium. 10 ml of this diluted fresh whole blood was then layered over 4.5 ml of Ficoll-Paque (FP) and centrifuged (450 × g, 25 °C ) for 30 min. The supernatant was aspirated down to approximately 1 cm above the Ficoll-Paque layer. The peripheral blood mononuclear cell (PBMC) layer, containing lymphocytes, was transferred to a 15 ml polypropylene tube which contained 10 ml RPMI with 5% FCS (25 °C ). Then the cells were centrifuged (15 min at 450 × g, 25 °C ) and the supernatant aspirated leaving a fi nal volume of 1ml; the lymphocytes were resuspended in 1 ml warm (37 °C ) Hanks' balanced salt solution (HBSS; 137 mM NaCl, 5.4 mM KCl, 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.4 mM KH 2 PO 4 , 0.4 mM MgSO 4 , 0.3 mM Na 2 HPO 4 , 25 mM Hepes, pH 7.4) and transferred to a 15 ml polypropylene tube. Cells were used for the measurements within 6 h.

Determination of the intracellular CP655 fl uorescence intensity
Freshly isolated lymphocytes were seeded at a density of 1.5 × 10 7 cells/ml on a glass coverslip that has been coated with poly-lysine (5 µg/cm 2 ) in a modifi ed Pentz chamber. After 10-15 min of incubation at 37 °C , the adherent cells were washed twice with HBSS and loaded with CP655 (30 µM; clogP of iron free ligand at 0.43 and of iron complex at 0.29) for 10 min in HBSS (37 °C ). Lymphocytes were then carefully washed three times with HBSS and 6 ml of fresh HBSS (37 °C ) remained within the chamber during measurements. The fl uorescence measurements were performed using an inverted microscope (Axiovert 135 TV, Zeiss, Oberkochen, Germany) equipped with the Attofluor imaging system (Atto Instruments, Rockville, MD). The CP655-loaded cells were excited at 425 ± 22.5 nm and the emission was monitored at 450-490 nm every 60 s using a bandpass fi lter. The exciting light was attenuated to 4% using gray fi lters in order to avoid photochemical damaging of the cells. The intracellular chelatable iron pool was manipulated 5 min after the beginning of the measurements by adding either the membrane-permeable iron(III)-8hydroxyquinoline complex (15 µM) that had been freshly prepared from stock solutions of ferric chloride (10 mM) and 8-hydroxyquinoline (8-HQ; 20 mM) in dimethyl sulfoxide (DMSO) or by the membranepermeable iron chelator, CP94 (1 mM; clogP of iron free ligand at 0.16 and of iron complex at -0.40), which had been freshly prepared by dissolving CP94 (100 mM) in 18 mΩ Millipore water. CP655 fl uorescence was then recorded until it stabilized (Fig. 1). The autofl uorescence from a parallel experiment with unloaded control lymphocytes as obtained using same instrument settings was set at 0% probe fl uorescence intensity. The fl uorescence intensity of the probeloaded lymphocytes at the end of the experiment with CP94 was set at 100% and used to calculate the relative initial CP655 fl uorescence intensity of the cells (Fig. 1). Since the measurements were performed on the single cell level, it was possible to differentiate lymphocytes from other PBMC, like monocytes. Solely lymphocytes were selected for evaluation.

Determination of the intracellular probe concentration
Cells were loaded with CP655 as described above. After washing the lymphocytes with HBSS, fl uorescence measurements were performed using a laser scanning microscope (LSM 510, Zeiss, Oberkochen, Germany) equipped with an argon laser. The intracellular fluorescence of CP655, after being fully dequenched by CP94 (1 mM), was excited at 458 nm and observed through a 475 nm bandpass fi lter. The fl uorescence measurements were performed in a focal plane 5-10 µm above the surface of the glass coverslip. At the end of the experimental procedures the uptake of the vital dye propidium iodide was routinely determined in order to detect loss of cell viability. The red fl uorescence of propidium iodide excited at 543 nm using the helium/neon laser was collected through a 560 nm long-pass fi lter; nuclear staining of few dead cells suggested that the lymphocytes studied were not proliferating. This focal plane and identical scanning parameters for cellular CP655 fl uorescence were used for scanning the cellular autofl uorescence as well as the fluorescence of a Chelex 100-treated tris(hydroxymethyl)amino-methane buffer (Trisbuffer, pH 7.4, 37 °C ) containing known concentrations of CP655. The intracellular concentration of CP655 in lymphocytes was determined from the difference in fl uorescence of CP655-loaded lymphocytes and unloaded cells (ΔF) compared with the fl uorescence of iron-free CP655 standards in the Trisbuffer.

Ex situ calibration for the determination of human lymphocyte LIP
The quenching effect of Fe(II) on CP655 fl uorescence in a medium designed to simulate the composition of the cytosol (37 o C) was determined using digital fl uorescence microscopy at the same settings as used for the cellular measurements (see above). This cytosolic medium contained 100 mM KCl, 5 mM Na 2 HPO 4 , 4 mM ATP, 2 mM MgCl 2 , 6.85 mM glucose, 0.138 mM pyruvate, 1.5 mM L-lactate, 0.23 mM sodium citrate, 2.99 mM potassium phosphate, the amino acid composition of Eagle's minimum essential medium, 2 mM ascorbic acid, 4.5 mM glutathione (GSH) and 10 mM imidazole, pH 7.2. Aliquots (2 ml) of the medium were placed on a glass coverslip within a modifi ed Pentz chamber, and CP655 at a fi nal concentration of 6 µM, i.e. the intracellular concentration of the probe (see below), was added and mixed with the medium. After the initial stable fl uorescence had been recorded, known concentrations of Fe(II) were added from a freshly prepared stock solution (0.5 mM ferrous ammonium sulfate plus 10 mM L-ascorbic acid). The addition of iron was continued until no additional fl uorescence quenching could be observed.

Results and Discussion
CP655 fl uorescence in lymphocytes could be both decreased and increased by the addition of iron and iron chelators, respectively. The addition of iron(III)-8-hydroxyquinoline (1:2) at a concentration of 15 µM almost completely quenched the intracellular probe fluorescence (Fig. 1). In controls, i.e. no addition of the iron complex, the  intracellular probe fl uorescence remained stable for at least 30 min. In other experiments, the addition of a large excess of the membrane-permeable iron chelator, CP94 (1 mM, fi nal concentration), signifi cantly enhanced the intracellular probe fl uorescence (Fig. 1). The intracellular probe fl uorescence in lymphocytes was found to be relatively weak and the probe dequenching to be relatively low when 10 µM probe was used for cellular loading. Therefore, we increased the CP655 loading concentration to 20, 30, 40 and 50 µM, respectively, and found that a maximum fl uorescence increase in lymphocytes was obtained when 30 µM of the probe had been used (data not shown). In contrast to low intracellular probe concentrations, which may be insuffi cient to completely chelate the cellular labile iron, high intracellular probe concentrations may promote probe leakage from the cells during the time-course of the measurements, rendering the system rather insensitive towards the detection of the small pool of intracellular chelatable iron. Thus, 30 µM CP655 was adopted as an optimal loading concentration for the determination of the LIP in lymphocytes. This loading concentration of CP655 did not infl uence the cell viability as judged by the vital dye, propidium iodide, which was added to the lymphocytes at the end of the experiments. When different CP655 concentrations (0-20 µM, iron-free probe) in a cell-free system were plotted versus their fl uorescence intensity as determined using laser scanning microscopy, the fl uorescence intensity was found to increase in a linearly proportional fashion with the concentration of the probe standards (data not shown). The total cellular fl uorescence (F total = CP655 fl uorescence + cellular autofl uorescence) was measured 5-10 min after the addition of an excess of CP94 (1 mM) to the supernatant (Fig. 2A). The difference (ΔF) between F total and the autofl uorescence of unloaded lymphocytes as determined at the same instrument setting (F auto ) (Fig. 2B) represented the intracellular CP655 fl uorescence (F CP655 ). Based on F CP655 and the above standard curve, a mean CP655 concentration of 5.9 ± 1.5 µM was determined in human lymphocytes.
CP655 at a concentration of 6 µM, i.e. the intracellular probe concentration, was used to perform an ex situ calibration procedure. Calibrations were performed using digital fl uorescence microscopy and a cytosolic medium (37 °C) which has been designed to simulate the cytosol composition (Fig. 3A). The fl uorescence signal of the iron-free probe (6 µM) was set at 100% probe fl uorescence intensity, the autofl uorescence of the cytosolic medium in the absence of CP655 was determined at the same instrument settings and set at 0% (Fig. 3B). Small amounts of iron (II) were added to the calibration medium and recordings continued until the progressive quenching of CP655 fl uorescence leveled off, suggesting that a steady state had been reached (Fig. 3A). Based on this ex situ calibration curve and relative intracellular fl uorescence intensity of the initial CP655 fl uorescence of 79 ± 8% (Fig. 1), the concentration of intracellular chelatable iron in lymphocytes was determined to be 0.57 ± 0.27 µM (n = 8). This value is well in line with the value of 0.53 ± 0.58 µM LIP reported for healthy human lymphocytes by Gackowski et al. (2002), and comparable to the levels that have been determined in monocytes (Epsztejn et al. 1997), but much less than the hepatocyte LIP (5.4 ± 1.3 µM) determined in our previous report (Ma et al. 2006). The fi nding that there is good agreement with the results reported by Gackowski et al. (2002) for lymphocytes, would indicate that, for this type of cell, the use of calcein provides a realistic estimate of LIP.
Previously, we have reported that intracellular ligands for iron like phosphates compete with low CP655 concentrations for iron binding (Ma et al. 2006). However, it in unlikely that these ligands strongly affected the intracellular determination of the lymphocyte LIP as reported here, since the ex situ calibrations were performed in a medium designed to simulate the composition of the cytosol, i.e. in a medium containing physiological concentrations of cellular ligands for iron.
In conclusion, in the present study we introduce an assay for the determination of the LIP in single intact lymphocytes based on a combination of digital fl uorescence microscopy and laser scanning microscopy. This assay depends on the specifi c quenching of CP655 by cellular chelatable iron. The method described herein has the advantages of simplicity, relatively low cost, high sensitivity and permits measurement at the single cell level. CP655, a novel fl uorescent iron indicator, has been found to be highly sensitive to the presence of intracellular chelatable iron and does not cause obvious cytotoxic effects. In contrast to calcein (Hasinoff, 2003), the indicator is not prone to oxidative damage in the presence of ferrous labile iron. These properties render it suit- able for monitoring the chelatable iron pool in various cell-types and therefore of providing information on the effi cacy of different chelators currently under investigation for treatment of iron overload. The fl uorescence intensity of CP655 (6 µM) as recorded using digital fl uorescence microscopy (λ ex = 425 ± 22.5 nm, λ em = 450-490 nm) in cytosolic medium was quenched by additions of iron (II) (0.3 µM each as shown by arrow) until the progressive quenching leveled to a steady state value. In addition, the fl uorescence of the cytosolic medium, without CP655, was recorded at the same instrument settings and used as a blank. Each trace shown is representative for the average of 3 experiments. (B) The fl uorescence intensity of iron-free CP655 (6 µM) was set at 100% relative fl uorescence intensity; 0% fl uorescence is equal to the fl uorescence of cytosolic medium without CP655. Values shown were obtained from Figure 3A.